Despite the importance of Clostridium botulinum as a human and animal pathogen, and its potential role in bioterrorism, the organism remains extremely poorly characterized with regard to cellular processes and their role in pathogenesis. This is almost entirely a consequence of ineffective genetic systems, and in particular integrational tools. This inability to easily generate stable mutants (directed or random) is common to all clostridial species, and severely hinders the ability of the scientific community at large to fully exploit clostridial genome information in hypothesis driven research. The goal of this project is specifically to develop the necessary enabling technology to overcome this impediment. The aims are: (1) to develop integrational vectors to introduce targeted mutations in genes of C. botulinum; (2) to identify effective transposons for random mutagenesis and (3) to assess antisense RNA strategies for modulation and analysis of C. botulinum gene expression. These aims will be accomplished through the complementary knowledge and expertise of a USA and UK laboratory. The developed genetic tools will pave the way for in depth analysis of not only the C. botulinum genome, but other clostridial genomes too. Their future deployment will, for example, lead to the elucidation of physiological factors that control growth and toxin production, pathogenesis, and developmental processes unique to clostridia and related organisms including endospore formation, resistance, germination and outgrowth.